It is often desirable to test a sample for reactivity against a multiplicity of reagents. For example, in cytokine secretion profiling, the ability of the secreted proteins from a particular tissue to immunoreact with a panel of antibodies raised with respect to the family of antigens is required, with tens to hundreds of family members of potential interest. In this instance, detection of the presence of the antibody-antigen complex normally requires that either a label be attached to the antibody in the complex or that a second antibody be bound to the first antibody where the second antibody has a label attached to it. Then, detection of the label confirms the presence of the antibody-antigen complex in the sample.
Commonly, the second antibody is a biotinylated goat anti-primary IgG that will react with avidin-horse radish peroxidase and, in the presence of a redox sensitive color indicator and substrate (hydrogen peroxide), result in a change in color, on a filter for example, indicating the presence of the antibody-antigen complex. Alternatively, instead of biotinylating the secondary antibody, an 125I-labeled secondary antibody can be used. If an 125I-label is used, exposure of the filter to X-ray film will allow for the detection of the antibody-antigen complex.
Often the signal emitted from these labels is not strong enough to be detected due to the low expression level of the protein of interest or limited supply of specimen. In addition, although it is possible to detect the presence of several different antigens in a sample by using an antibody directed towards each antigen, a common readout for all antibodies makes it impossible to clearly distinguish one antigen-antibody complex from another without prior fractionation (e.g., by gel electrophoresis) or parallel assay of specimen aliquots (including capture on discrete locations on a chip or a combinatorially colored particle set). To address the sensitivity issue, it would be advantageous to have a label that emits a signal strong enough to allow detection of antigens in a sample even if the antigen is present at low amounts. It would further be advantageous to determine the presence of multiple antigens in a complex mixture by virtue of a family of such labels with distinguishable signals. For example, serological tissue typing for HLA antigens probes ˜6 genetic loci for dozens to hundreds of allelic variants at each locus. Any given individual will at most express 2 alleles for each locus, but hundreds of separate assays are needed to accomplish the typing. With multiplexing, all the assays can be run on one specimen, providing a more efficient system. For an application such as this, it is important that the staining reagents themselves be multiplexed, as contrasted to multiplexed binding surfaces to which a single staining reagent binds.
An even more compelling need for sensitive, highly multiplexed detection is readily apparent in the case of cytokine secretion assays in which the proteins secreted from a single cell are captured on the underlying surface and then analyzed in situ. Such assays have heretofore only been described at the 2-plex level, with the vast majority of work at the 1-plex level to avoid the increased assay complexity inherent in previously available multiplexing approaches. Increasing the multiplexing capacity enables identification of novel T-cell subtypes that would require immense effort to discover by looking only at pairwise combinations of the two dozen or more cytokines. More generally, normal cell to cell variation makes it difficult to identify novel cell types based on multivariate properties when only two or three properties are measured per cell.
It is also desirable to multiplex DNA sequencing. Originally, sequencing by the chain-termination method involves the synthesis of a DNA strand by a DNA polymerase using a single stranded template. Synthesis initiated at the site where an oligonucleotide primer anneals to the template was terminated by the incorporation of a low level of radio-labeled nucleotide analog (ddNTP) into the elongation reagent cocktail. When proper mixtures of dNTP's and one of the four ddNTP's are used, polymerization terminates randomly at each possible site allowing for the sequence of the DNA to be read following size separation by gel or capillary electrophoresis. Four parallel reactions are fractionated in parallel lanes to identify the base at each termination length. More recently, the four reactions have each been terminated using a ddNTP conjugated to a different color of fluorescent dye, and all four reaction product mixtures fractionated together in one lane of a gel or one capillary electrophoresis channel. Thus, what was originally a 1-plex assay became a 4-plex assay. With the additional multiplexing capacity of the present invention, more than one DNA template could be sequenced in a single lane or capillary.
High detection sensitivity is also important for multiplexed DNA sequencing to avoid overloading the gel or capillary which can perturb the migration of the DNA molecules. The signal emitted from conventional radioactive or fluorescent dye-based labels is often not strong enough to be detected without extensive amplification of the DNA; a more sensitive label enables decreasing assay complexity and the associated potential for artifactual results. And, with the extra data channels provided by sensitive multiplexing, an internal sizing ladder can be included in every lane.
One embodiment of suitable multiplexing technology has been previously described in detail in U.S. Pat. Nos. 6,642,062 and 6,492,125. Briefly, in a preferred embodiment latex (polystyrene) particles are impregnated with organic dye fluors in varying ratios to generate a combinatorially coded set of labels. Alternatively, fluorescent labels comprising nanocrystalline semiconductor structures of various types, commonly called quantum dots, may be employed. Such particles can also be coupled to biorecognition molecules, or can be used in other particulates in varied ratios as substitutes for fluorescent dyes. Han, M. Y., et al., Nat. Biotechnol. (2001) 19:631-635 describes polystyrene particles embedded with multicolor CdSe quantum dots at various color and intensity combinations. Based on entirely different principles, Nicewarner-Pena, S. R., et al., Science (2001) 294:137-141 have reported a metallic nanobarcoding technology for multiplexed bioassays.
The use of submicron particles that are bright enough for single particle detection enables a variety of assay formats not accessible to conventional signal generating labels for which integrated intensity of a population of labels is measured. In addition to latex microspheres and quantum dots, for which fluorescence is the signal, other possible signals include phosphorescence, NMR spectra and Raman spectra, and modifiable reflectance properties.
The use of particulate labels to investigate spatial relationships among individual cellular components is described in U.S. Pat. No. 6,642,062, incorporated herein by reference. As described in this patent, individual particles coupled to reagents specific for various cellular components can be prepared in a multiplicity of distinguishable “hues” which are detectable by microscopy and can provide a picture of the spatial arrangement of intracellular components and organelles. Further, as described in U.S. Pat. No. 6,673,554, the changes in spatial arrangement of these components in response to stimuli may be used to evaluate the toxicity of compounds and to identify treatment protocols for disease conditions. The present invention, in one embodiment, relates specifically to the application of these techniques to clinical biopsy samples using these and additional techniques which rely on the sensitivity and multivariant nature of particulate labeling. Certain improvements in particulate labels themselves are also described.
The identity of the particulate labels can be assessed, e.g., for fluorescent labels with a suitable excitation light source and emission filters able to detect wavelengths from the blue to the near infrared, microscopically to determine the position or presence of a single particulate label. Therefore, multiple antigen-antibody complexes, or other biospecific pairs, can be distinguished in a sample by the unique emission properties of each particulate label, enabling multiple parallel assays to be run at the same time in the same physical chamber. In addition, due to the high detectability of particulate labels, as compared to conventional dye molecules, sequencing reactions and the like can be run even when available sample size is too small for conventional analysis. Thus the present invention provides for a more sensitive and more versatile approach to detect the presence of proteins, nucleic acids and the like in a sample or samples. Specific innovative uses of particulate labels are disclosed relating to particular types of multiplexed biospecific interactions. Certain of these assays, whose development was prompted by the availability of convenient particulate labels, are novel in their own right. Although particulate labels provide a preferred embodiment, the use of particulate labels is not a strict requirement for such assays.
Assays of clinical interest are also provided. It is well understood that the cost of bringing a new drug to market is now of the order of $800,000,000, a number driven by the high failure rate of drug candidates. Most of this failure rate is attributable to the reliance by the industry on animal studies in preclinical trials; the transition from results in animals to results in humans is not marked by a one-to-one correspondence. It would therefore represent a step forward to utilize biopsied human tissue samples to assess disease conditions and efficacy of drugs. The present invention facilitates the use of such samples.